Conference posters, 2010 TIMBR

Chemistry and Kinetics of Furan Conversion into Aromatics and Olefins over ZSM-5: A Model Biomass Conversion Reaction

Yu-Ting Cheng et al.

The conversion of furan (a model of cellulosic biomass) over ZSM-5 was studied in a TGA/TPD-MS system, an in-situ FTIR, and in a continuous flow fixed-bed reactor. The furan adsorbs as oligomers at room temperature with a 1.73 of adsorbed furan/Al ratio. These oligomers are converted to CO, CO2, olefins and aromatics at temperatures from 400 – 600 ºC. We have measured the effects of space velocity, temperature, and partial pressure for furan conversion to help us understand the chemistry of biomass conversion inside zeolite catalysts. The selectivity of aromatics and olefins products did not change significantly with space velocity. The apparent reaction order for furan consumption with respect to furan was lower (1.2) than the apparent reaction orders for aromatics (1.7) and olefins formation (1.5). The apparent activation energies of furan consumption (26 kJ/mol) and coke formation (22 kJ/mol) were lower than the apparent activation energies for formation of olefins and aromatics (50-60 kJ/mol). Coke deposition was fast and dramatically deactivated the catalyst in 1 hour. Kinetic data obtained in this study is strongly pore-diffusion controlled and is far from thermodynamic equilibrium. We have proposed some key elementary reactions that may occur for this process.

A Protein-DNA Interaction Network For Cell Wall Biosynthesis

Li Lin et al.

Plant cell walls comprise a complex of mostly cellulose, hemicellulose, and lignin. The composition and interaction among these three constituents dictate in large part the amenability of plant feedstocks for conversion to simple sugars and then to biofuels. One mechanism regulating cell wall biosynthesis is the activity of transcription factors that control higher order events of growth and differentiation; the likely direct regulation of processive and non- processive glycosyltransferases as well as the phenylpropanoid metabolic grid. We measured interactions among the promoters of Arabidopsis cell wall genes and a nearly comprehensive transcription factor library using a high throughput yeast one-hybrid assay. In addition to several NAC and numerous MYB transcription factors some of which have been implicated in cell wall regulation, we measured interactions among twenty-three other families and cell wall promoters. While most (85%) bind just one, some bind multiple promoters. Interestingly, cellulose, hemicellulose, and lignin cis-regulatory regions share several interactors. A loss-of-function mutant of one such gene exhibits significantly more vascular bundles, which make up an appreciable proportion of overall plant biomass. With further screens for protein-DNA interactions, we will resolve either interlocking or independent networks leading to synthesis of cellulose, hemicellulose, and lignin. Ultimately, we hope to determine the effects of regulator perturbation on amenability to deconstruction and cell wall properties.

Development of a high throughput translational bioassay for plant biofuel properties

Scott J. Lee et al.

Using the well-developed microbial system, Clostridium phytofermentans, we have developed a robust bioassay for biomass digestibility and conversion to biofuels. The bioassay can be used to measure the impact of plant genetic diversity on digestibility, and thereby determine the potential effects of altered energy crop traits. Moreover, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be critical in single stage fermentation or consolidated bioprocessing. In order to develop a baseline for our bioassay, we utilized two well characterized lignin mutants of sorghum, brown midrib-6 (bmr-6) and brown midrib-12 (bmr-12) and the double mutant (bmr-6/bmr-12). Lignin, a component of secondary cell walls, is strongly associated with plant tissue recalcitrance to conversion to biofuels. These mutants exhibit a significant reduction in total lignin content and are therefore more digestible. Whole, field grown, de-grained plants were ground to a fine powder and used as a substrate for C. phytofermentans growth. We detected significant differences in ethanol production among the sorghum genotypes by HPLC analysis of three day old anaerobic cultures. We also measured significant variation among different accessions of Arabidopsis thaliana and Brachypodium distachyon. By using C. phytofermentans as an indicator of feedstock quality we can observe differences both within and among species, as well as take into account specific plant-microbe interactions. Ultimately, we will use this assay to study the genetics of plant biofuel properties.

Production of Green Aromatics and Olefins by Catalytic Fast Pyrolysis of Wood

Torren R. Carlson et al.

Catalytic fast pyrolysis (CFP) is a promising process for the direct conversion of solid biomass into gasoline range aromatics. This novel process has significant advantages compared to other technologies for biomass conversion including low capital and operating costs and it makes a product that already fits into existing infrastructure. The CFP of pine wood and furan with ZSM-5 catalyst was studied under different reaction conditions with several different reactors including a fluidized bed reactor, a fixed bed reactor and a semi-batch pyroprobe reactor to optimize CFP for aromatic production. The highest aromatic yield of 14 % carbon was obtained at low space velocity and 600 oC. The aromatic product consists mainly of benzene (24.8 % carbon), toluene (34.1% carbon), xylene (15.4% carbon) and naphthalene (14.9 % carbon).

The aromatic yield and selectivity is a function of reactor temperature. However, the olefin yield was not a function of temperature. The selectivity for benzene and naphthalene increases at temperature increases. The more valuable aromatics toluene and xylene are selectively produced at lower temperature. We also studied furan conversion in a fixed bed reactor to help identify the catalytic chemistry. Our results from the fixed bed indicate that furan is a good model compound to study CFP with wood. The maximum aromatic yield (24% carbon) from furan was obtained at 600 °C which is consistent with the fluidized bed results.

Olefins can be recycled to the reactor inlet to produce more aromatics. Co-feeding olefins with wood increases both the aromatic yield and conversion of feed. With co-feed the selectivity of small aromatics (such as toluene and benzene) increases while the selectivity for naphthalenes decreases. In this poster presentation we estimate the aromatic yields that could be achieved by CFP when we include olefin recycle.

Optimizing the Shape Selectivity of Zeolite Catalysts for Biomass Conversion: The Kinetic Diameter

Jungho Jae et al.

We have studied the influence of catalyst pore size and morphology on the conversion of glucose to aromatics by catalytic fast pyrolysis using over 15 different zeolite catalysts having a variety of shapes and pore sizes. The estimated kinetic diameter for the catalytic pyrolysis products and reactants was used to determine the optimal pore size for zeolite catalysts for catalytic fast pyrolysis. Smaller oxygenate pyrolysis products including furans, hydroxyaldehydes, and organic acids are sufficiently small in diameter to diffuse easily into ZSM-5 (6.3 Å). Of the aromatic products only benzene, toluene, indane, indene, naphthalene, ethylbenzene and xylenes are of a sufficiently small size compared to the ZSM-5 pore. Zeolites type catalysts with a range of pore size 3.9-7.4Å were used for catalytic testing. From these an optimum pore size range of 5.7-6.6Å is identified to maximize aromatic yield. In addition to pore window size, zeolite pore structure and intersections are critical for the reaction mechanism. It is likely that this small pore size also limits the formation of larger aromatics including coke in the pores. Key words: Zeolite, Catalytic Fast Pyrolysis, Kinetic Diameter, Aromatics.

IAA Biosynthesis in Brachypodium distachyon

Caroline Duffy et al.

The grass Brachypodium distachyon has been identified as a model system to study energy crops for the production of cellulosic ethanol. Brachypodium’s small genome has recently been fully sequenced to further understand its biology. By profiling the auxin, indole-3-acetic acid (IAA), in Brachypodium, we are trying to establish growth conditions in which we can detect quantifiable differences in IAA levels. Comparing IAA levels with transcript levels for putative Brachypodium orthologs (functionally similar genes from difference species) to predicted Arabidopsis thaliana IAA biosynthetic genes will then provide evidence for or against specific Brachypodium genes being involved in IAA synthesis.

The Brachypodium genome database has been mined using the BLAST bioinformatics algorithm to identify potential orthologs to a number of Arabidopsis IAA biosynthetic genes. Brachypodium wildtype tissue has been grown under full and etiolated (dark) light conditions, and IAA levels determined at various stages of root and shoot development through the use of solid phase extraction (SPE) and gas chromatograph-mass spectrometry (GC-MS) methods. Preliminary results show differences in IAA levels between root and shoot tissue. Previous work on IAA biosynthetic pathways in Arabidopsis shows that IAA is derived from the amino acid tryptophan (Trp) during early stages of development. With these tentative differences in mind, the goals of this project will focus on two research objectives. First, pulse-labeling experiments using stable-isotope labeled Trp and anthranilate will be conducted to determine if a similar Trp-dependent pathway is observed in Brachypodium. Second, once significant differences between growth conditions and IAA levels are observed, RNA from Brachypodium will be extracted, converted to cDNA via reverse transcription and applied to a microarray chip containing the Brachypodium genome to quantify differences in transcript expression between the two growth conditions in order to identify genes potentially involved in IAA biosynthesis.

Dynamic Flux Balance Modeling of a Microbial Co-Culture for Efficient Batch Fermentation of Glucose and Xylose Mixtures

Timothy J. Hanly et al.

A requirement for the economically viable production of fuels from cellulosic biomass is the efficient consumption and conversion of its constituent sugars. Genetically engineering a single organism to metabolize multiple sugars typically results in inefficiencies due to diauxic growth and/or limitations in substrate uptakes. We present a mathematical model of a synthetic consortium for the production of ethanol from mixtures of glucose and xylose. The co-culture is composed of wild-type Saccharomyces cerevisiae and Escherichia coli strain ZSC113, two microbes that will specifically uptake glucose and xylose, respectively. Dynamic flux balance analysis is employed to compare this co-culture to mono-cultures of individual S. cerevisiae and E. coli strains capable of consuming both sugars with respect to their batch ethanol productivities. The effects of altering the amount of each microbe present in reactor inoculum and changing the time at which the batch is switched from aerobic to anaerobic cultivation are investigated. Through these process engineering strategies, a nearly two-fold increase in ethanol productivity over pure cultures is predicted under the assumption of optimal growth conditions for each microbe. Future work will focus on verifying these results experimentally to test the limitations of this assumption and the assumption that the microbes are non-interacting.

The Intrinsic Kinetics and Heats of Reactions for Cellulose Pyrolysis and Char Formation

Joungmo Cho et al.

The conversion of biomass into biofuel products by pyrolysis has attracted tremendous interests due to high availability and potential to provide sustainable liquid fuels. Cellulose is the most abundant polymeric carbohydrate compound, which comprise the largest fraction of biomass. During the pyrolysis, cellulose undergoes multiple decomposition pathways depending on the thermal conditions and produces different classes of compounds including non-condensible gases, condensable vapors that are condensed into a liquid mixture, solid chars. In spite of numerous efforts to understand reaction mechanism for cellulose pyrolysis, a reliable reaction model describing both heat release and intrinsic themokinetic behavior is not developed yet due to the lack of means to quantify the heat involvement of individual steps. In the present study, cellulose pyrolysis experiments at isothermal and dynamic conditions are carried out to characterize the evolution of products depending on thermal conditions. Observed decomposition behaviors are compared with an intrinsic kinetic model to estimate the reaction rates and heats of reactions for individual reaction steps.

Non-coding RNA Control of Plant Cell Wall Biosynthesis and Biofuel Properties

Rebecca C. Lamothe et al.

The synthesis of secondary cell walls is an important process in vascular plants. Walls not only make up the vascular system necessary to transport water and nutrients, they also act as a barrier against various predators and other environmental insults. While these abilities are vital to the survival of plants, they also make them difficult to process as feedstock for biofuel production. There are many genes that regulate the secondary cell wall biosynthesis, particularly the formation of one of its components: lignin, including microRNAs (miRNAs). As their name implies, miRNAs are short RNA transcripts; however, unlike mRNAs, they are not translated into proteins, but play critical roles in gene regulation through their interactions with other transcripts. In the model plant Arabidopsis, miRNA are predicted to play a role in the degradation cell wall biosynthesis genes. In order to functionally characterize these miRNA, they were cloned into a series of vectors and transformed using the bacterium Agrobacterium tumefacians into Arabidopsis. We will characterize target gene expression as well as the cell wall phenotype of mutant plants, including their biofuel feedstock quality. We will also screen for transcription factor proteins that interact with the miRNA promoters using a yeast one-hybrid assay. If the miRNA do indeed play a role in cell wall transcript stability, there should be an observable vascular development defect and altered cell wall properties perhaps resulting in a plant more amenable to conversion to biofuels.

Insight into grass cell wall biosynthesis

Pubudu Handakumbura

Insight into grass cell wall biosynthesis

Pubudu Handakumbura, Samuel P Hazen

Department of Biology, University of Massachusetts, Amherst, MA

Utilization of domestic resources has become a necessity in order to maximize the potential for sufficient energy production. Biofuels are a promising renewable energy source that has great potential to meet global energy needs. Cellulose, one of the key components of the plant cell wall, is the most abundant, potentially cost-effective and renewable carbon source available on earth. This polysaccharide can be readily converted into simple sugars suitable for biofuel conversion. A major limiting factor to using plant biomass is the cost associated with feedstock pretreatment that is required to deconstruct biomass to simple sugars. We seek to develop energy crops that are highly amenable to biomass feedstock conversion through elucidating and manipulating cell wall biosynthesis of grasses. We have generated loss-of-function mutants in the grass model specie Brachypodium distachyon by specifically targeting putative cell wall genes using artificial microRNAs. We are also developing mutants of putative key transcriptional regulators in order to elucidate the regulation of cell wall biosynthesis. The resulting transgenic plants will be analyzed for changes in target gene expression, cell wall morphology, and biofuel feedstock properties. Functional characterization of these key biosynthetic enzymes and transcription factors will provide a starting point to understanding the transcriptional regulation and the potential for energy crop improvement.